Inhibition of tace or amphiregulin for the modulation of egf receptor signal transactivation

ABSTRACT

The present invention relates to the modulation of transactivation of receptor tyrosine kinases by G protein or G protein-coupled receptor (GPCR) mediated signal transduction in a cell or an organism comprising inhibiting the activity of the metalloprotease TACE/ADAM17 and/or the activity of the receptor tyrosine kinase ligand amphiregulin.

The present invention relates to the modulation of transactivation ofreceptor tyrosine kinases by G protein or G protein-coupled receptor(GPCR) mediated signal transduction in a cell or an organism comprisinginhibiting the activity of the metalloprotease TACE/ADAM17 and/or theactivity of the receptor tyrosine kinase ligand amphiregulin.

Communication between G protein-coupled receptor (GPCR) and EGFRsignalling systems involves cell surface proteolysis of the growthfactor precursor proHB-EGF (1-3). The molecular mechanism of EGFR signaltransactivation in human cancer cells, however, is largely unknown.

Interreceptor communication between G protein-coupled receptors (GPCRs)and the EGFR occurs in diverse cell types including fibroblasts,keratinocytes and smooth muscle cells (4). Treatment of cells with GPCRagonists results in activation and tyrosine phosphorylation of the EGFRand subsequently leads to the generation of an EGFR-characteristic,intracellular signal (5). Due to the rapid kinetics of the EGFRtransactivation signal and the fact that release of EGFR ligands was notdetectable after GPCR stimulation, the mechanism of EGFR transactivationwas proposed to exclusively rely on intracellular elements (5, 6). Incontrast, a novel mechanistic concept of EGFR transactivation involvesthe proteolytic release of heparin-binding EGF-like growth factor(HB-EGF) at the cell surface of GPCR stimulated cells (1). HB-EGF, aswell as transforming growth factor alpha (TGFa) and amphiregulin (AR)belong to a family of EGF-like ligands that directly activate the EGFR.These molecules are synthesized as transmembrane precursors and aresubject to proteolytic cleavage to produce the soluble and diffusiblegrowth factors (7). The HB-EGF-dependent mechanism of EGFR signaltransactivation has gained further experimental support by studies onGPCR mitogenic signalling in vascular smooth muscle cells (8), cardiacendothelial cells (9) and cardiomyocytes (10). Importantly, recent datahave implicated EGFR signal transactivation pathways in the etiology ofpathobiological processes such as cystic fibrosis (3), cardiac (2) andgastrointestinal hypertrophy (11). Furthermore, increasing evidenceargues for a direct correlation between aberrant GPCR signalling and thedevelopment and progression of human cancers (12). We have recentlydemonstrated that GPCR-EGFR cross-talk pathways are widely establishedin head and neck squamous cell carcinoma (HNSCC) cells and that GPCRagonists such as LPA and carbachol regulate the proliferative andmigratory behavior of HNSCC cells via transactivation of the EGFR (13).Elucidation of the molecular mechanisms underlying EGFR signaltransactivation may thus lead to new strategies for the prevention andtreatment of carcinomas, e.g. squamous cell carcinomas.

Here, we demonstrate that in squamous cell carcinoma cells stimulationwith the GPCR agonists lysophosphatidic acid (LPA) or carbacholspecifically results in metalloprotease-dependent cleavage and releaseof the EGFR ligand amphiregulin (AR). Moreover, AR gene silencing bysmall interfering RNA (siRNA) or inhibition of AR biological activity byneutralizing antibodies prevents GPCR-induced EGFR tyrosinephosphorylation, downstream mitogenic signalling events, activation ofAkt/PKB, cell proliferation and migration. Furthermore, we presentevidence that in squamous cell carcinoma cells blockade of themetalloprotease-disintegrin TACE/ADAM17 by expression of a dominantnegative mutant or by RNA interference suppresses GPCR stimulated ARrelease and EGFR-dependent cellular responses. Thus, TACE and/or AR canfunction as an effector of GPCR-mediated signalling and thereforerepresents a key element of the cellular receptor cross-talk network.

In a first aspect, the invention relates to a method for modulatingtransactivation of receptor tyrosine kinases by G protein or Gprotein-coupled receptor mediated signal transduction in a cellcomprising inhibiting the activity of the metalloprotease TACE/ADAM17and/or the activity of the receptor tyrosine kinase ligand amphiregulin.

The term “inhibition” according to the present invention preferablyrelates to a “specific” inhibition, wherein the activity of TACE/ADAM17and/or amphiregulin is selectively inhibited, i.e. the activity of othermetalloproteases such as ADAM12 or other receptor tyrosine kinaseligands such as HB-EGF is not significantly inhibited. By means ofselective inhibition of TACE/ADAM17 and/or amphiregulin a highlyspecific disruption of receptor tyrosine kinase transactivation may beachieved which is important for pharmaceutical applications in that theoccurance of undesired side effects may be reduced.

Further, the term “inhibition” preferably relates to a “direct”inhibition, wherein the inhibitor directly binds to TACE/ADAM17 and/oramphiregulin or a nucleic acid molecule coding therefor. The invention,however, also encompasses an “indirect” inhibtion wherein the inhibitordoes not directly bind to TACE/ADAM17 and/or amphiregulin but to aprecursor or metabolite thereof, particularly the amphiregulin precursorproamphiregulin.

The term “activity” preferably relates to the cleavage ofproamphiregulin by TACE/ADAM17 and/or the activation of a receptortyrosine kinase, e.g. EGFR by amphiregulin. A TACE/ADAM17 inhibitor ofthe present invention is preferably capable of inhibiting the cleavageand release of the receptor tyrosine kinase ligand amphiregulin. Anamphiregulin inhibitor of the present invention is preferably capable ofinhibiting biological activity of amphiregulin, particularly EGFRtyrosine phosphorylation, downstream mitogenic signaling events,activation of Akt/PKB, cell proliferation and/or migration.

A further aspect of the present invention is the use of an inhibitor ofthe metalloprotease TACE/ADAM17 and/or an inhibitor of the receptortyrosine kinase ligand amphiregulin for the prevention and/or treatmentof a disorder which is caused by or associated with a transactivation ofreceptor tyrosine kinases by G protein oder G protein-coupled receptormediated signal transduction. The presence of such a type of disordermay be determined by measuring G protein and/or GPCR expression, e.g. onthe mRNA level (cDNA array analysis, SAGE, Northern blot, etc.) and/oron the protein level (Western blot analysis, ImmunofluorescenceMicroscopy, in situ hybridisation techniques, etc.). The presence ofsuch a type of disorder may also be determined by examining theoccurrence of activating mutations in genomic and/or mRNA moleculesencoding G proteins or GPCRs and/or the presence of virally encodedGPCRs. Further, elevated levels of GPCR agonists such as LPA and/oramphiregulin in serum and/or disease-affected tissues may be determined.It should be pointed out that this type of disorder need not beassociated with enhanced receptor tyrosine kinase expression.

For example, the disorder may be a hyperproliferative disorder such ascancer, e.g. squamous cell carcinoma or another disorder such as ahyperproliferative skin disease, e.g. psoriasis.

The activity of TACE/ADAM17 and/or amphiregulin may be inhibited on thenucleic acid level, e.g. on the gene level or on the transcriptionlevel. Inhibition on the gene level may comprise a partial or completegene inactivation, i.e. by gene disruption. On the other hand,inhibition may occur on the transcript level, e.g. by application ofantisense molecules, e.g. DNA molecules, RNA molecules or nucleic acidanalogues, ribozymes, e.g. RNA molecules or nucleic acid analogues orsmall RNA molecules capable of RNA interference (RNAi), e.g. RNAmolecules or nucleic acid analogues, directed against TACE/ADAM17 and/oramphiregulin mRNA. Antisense molecules inhibiting the expression ofTACE/ADAM17 are for example described in U.S. Pat. No. 6,180,403, whichis herein incorporated by reference.

Further, the activity of TACE/ADAM17 and/or amphiregulin may beinhibited on the protein level, e.g. by application of compounds whichresult in a specific inhibition of TACE/ADAM17 and/or amphiregulinacitivity. The inhbition on the protein level may comprise for examplethe application of antibodies or antibody fragments directed againstTACE/ADAM17 and/or amphiregulin. The antibodies may be polyclonalantibodies or monoclonal antibodies, recombinant antibodies, e.g. singlechain antibodies or fragments of such antibodies which contain at leastone antigen-binding site, e.g. proteolytic antibody fragments such asFab, Fab′ or F(ab′)2 fragments or recombinant antibody fragments such asscFv fragments. For therapeutic purposes, particularly for the treatmentof humans, the application of chimeric antibodies, humanized antibodiesor human antibodies is especially preferred.

The antibodies or antibody fragments may be directed against themetalloprotease-domain of TACE/ADAM17, or against other parts of themolecule. The antibodies or antibody fragments may selectively recognizethe mature form of TACE/ADAM17, or the pro-form of TACE/ADAM17 as shownby immunoprecipitation. Alternatively, the antibodies or antibodyfragments may recognize both the mature form and the pro-form ofTACE/ADAM17.

Monoclonal antibodies may be generated by known techniques, e.g.hybridoma techniques as described by Köhler et al. (Nature 256 (1975),495-497), Cole et al. (Mol. Cell. Biol. 62 (1984), 109-120) or Kozbor etal. (J. Immunol. Meth. 81 (1985), 31-42) which are herein incorporatedby reference. Chimeric or humanized antibodies may be generated bytechniques described by Morrison et al. (Proc. Natl. Acad. Sci. USA 81(1984), 6851-6855), Neuberger et al. (Nature 312 (1984), 604-608),Takeda et al. (Nature 314 (1985), 452-454), Jones et al. (Nature 321(1986), 522-525), Riechmann et al. (Nature 322 (1988), 323-327),Verhoeyen et al. (Science 239 (1988), 1534-1536) or Queen et al. (Proc.Natl. Acad. Sci. USA 86 (1989), 10029-10033), which are hereinincorporated by reference. Further methods for generating antibodies orantibody fragments are described by Burton (Proc. Natl. Acad. Sci. USA88 (1991), 11120-11123), Orlandi et al., (Proc. Natl. Acad. Sci. USA 86(1989), 3833-3837), Winter et al. (Nature 349 (1991), 293-299) or Huseet al. (Science 254 (1989), 1275-1281), which are herein incorporated byreference.

Furthermore, low-molecular weight inhibitors of TACE/ADAM17 and/oramphiregulin may be used. Examples of TACE/ADAM17 inhibitors aresulfonic acid or phosphinic acid derivatives, e.g. sulfonamides,sulfonamide hydroxamic acids, phosphinic acid amide hydroxamic acids,e.g. as described in WO 98/16503, WO 98/16506, WO 98/16514, WO 98/16520,Mac Pherson et al. (J. Med. Chem. 40, (1997), 2525), Tamura et al. (J.Med. Chem. 41 (1998), 690), Levin et al. (Bioorg. & Med. Chem. Lett. 8(1998), 2657), Pikul et al. (J. Med. Chem. 41 (1998), 3568), WO97/18194, EP-A-0803505, WO 98/08853, WO 98/03166 and EP-A-1279674, whichare herein incorporated by reference. Further inhibitors may beidentified by screening procedures as outlined in detail below.

For therapeutic purposes, the medicament is administered in the form ofa pharmaceutical composition which additionally comprisespharmaceutically acceptable carriers, diluents and/or adjuvants.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. A therapeuticallyeffective dose refers to that amount of the compound that results inamelioration of symptoms or a prolongation of survival in a patient.Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures In cell cultures or experimentalanimals, e.g. for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). For any compound used in the method of the invention,the therapeutically effective dose can be estimated initially from cellculture assays. For example, a dose can be formulated in animal modelsto achieve a circulating concentration range that includes the IC50 asdetermined in cell culture (i.e. the concentration of the test compoundwhich achieves a half-maximal inhibition of the growth-factor receptoractivity). Such information can be used to more accurately determineuseful doses in humans. The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratiobetween LD50 and ED50. Compounds which exhibit high therapeutic indicesare preferred. The exact formulation, route of administration and dosagecan be chosen by the individual physician in view of the patient'scondition (see e.g. Fingl et al., 1975, in “The Pharmacological Basis ofTherapeutics”, Ch. 1, p. 1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety which are sufficient to maintain thereceptor modulating effects, or minimal effective concentration (MEC).The MEC will vary for each compound but can be estimated from in vitrodata, e.g. the concentration necessary to achieve a 50-90% inhibition ofthe receptor using the assays described herein. Compounds should beadministered using a regimen which maintains plasma levels above the MECfor 10-90% of the time, preferably between 30-90% and most preferablybetween 50-90%. Dosages necessary to achieve the MEC will depend onindividual characteristics and route of administration. In cases oflocal administration or selective uptake, the effective localconcentration of the drug may not be related to plasma concentration.

The actual amount of composition administered will, of course, bedependent on the subject being treated, on the subject's weight, theseverity of the affliction, the manner of administration and thejudgement of the prescribing physician. For antibodies ortherapeutically active nucleic acid molecules, and other compounds e.g.a daily dosage of 0.001 to 100 mg/kg, particularly 0.01 to 10 mg/kg perday is suitable.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections.

Alternatively, one may administer the compound in a local rather than asystematic manner, for example, via injection of the compound directlyinto a solid tumor, often in a depot or sustained release formulation.

Furthermore, one may administer the drug in a targeted drug deliverysystem, for example in a liposome coated with a tumor-specific antibody.The liposomes will be targeted to and taken up selectively by the tumor.

Still a further aspect of the present invention is a method foridentifying modulators of receptor tyrosine kinase transactivation by Gprotein or G protein-coupled receptor mediated signal transduction,comprising determining, if a test compound is capable of inhibiting theactivity of TACE/ADAM17 and/or the activity of amphiregulin. This methodis suitable as a screening procedure, e.g. a high-throughput screeningprocedure for identifying novel compounds or classes of compounds whichare capable of modulating G protein signal transduction. Further, themethod is suitable as a validation procedure for characterizing thepharmaceutical efficacy and/or the side effects of compounds. The methodmay comprise the use of isolated proteins, cell extracts, recombinantcells or transgenic non-human animals. The recombinant cells ortransgenic non-human animals preferably exhibit an altered TACE/ADAM17and/or amphiregulin expression compared to a corresponding wild-typecell or animal.

Examples of suitable receptor tyrosine kinases are EGFR and othermembers of the EGFR family such as HER2, HER3 or HER4, PDGFR, thevascular endothelial growth factor receptor KDR/Flk-1, the Trk receptor,FGFR-1 or IGF-1 receptor but also other types of growth-factor receptorssuch as TNF receptor 1, TNF receptor 2, CD30 and IL-6 receptor aretargets for the G protein/GPCR mediated signal transduction.

Furthermore, the invention should be explained by the following Figuresand Examples.

FIG. 1 GPCR stimulation of the EGFR Involves a ligand-dependentmechanism and is accompanied by AR release from the cell surface. a,EGFR signal transactivation requires metalloprotease activity and theEGFR extracellular domain. SCC-9 cells were pre-incubated withmarimastat (BB2516, 10 μM; 20 min), anti-EGFR antibody ICR-3R (20 μg/mL;60 min) or PTX (100 ng/mL; 18 h) and treated with LPA (10 μM), carbachol(Car, 1 mM), EGF (7.5 ng/mL) or pervanadate (PV, 1 mM) for 3 min.Following immunoprecipitation (IP) of cell extracts with anti-EGFRantibody proteins were immunoblotted (IB) with anti-phosphotyrosineantibody and re-probed with anti-EGFR antibody. b, Flow cytometricanalysis of EGF-like precursor expression. SCC-9 cells were collectedand stained for surface HB-EGF, TGFa or AR and analyzed by flowcytometry. Control cells were labelled with FITC-conjugated secondaryantibody alone. c, LPA-induced proteolytic processing of proAR. SCC-9cells were pre-incubated with batimastat (BB94, 10 μM) or PTX andstimulated with LPA or TPA (1 μM) for 5 min. Cells were collected andanalyzed for cell surface AR density by flow cytometry. d, GPCR-inducedproteolytic release of AR. SCC-9 cells were pre-incubated withbatimastat or vehicle followed by stimulation with agonists as indicatedfor 120 min. Conditioned medium was collected and analyzed for total ARamount by ELISA. Each bar is the average of triplicate values(mean±s.d.). *, P<0.03 for the difference between agonists vs.BB94+agonists.

FIG. 2 GPCR stimulation requires AR to trigger EGFR-dependent signalsand biological responses. a, Blockade of EGF-like growth factorprecursor expression by RNA interference (RNAi). SCC-9 cells weretransfected with siRNA for proAR, proHB-EGF or proTGFα cultured for 2days and analyzed for gene expression by RT-PCR as indicated or b,stimulated with LPA or carbachol and assayed for EGFR tyrosinephosphorylation content. c, Requirement of AR for LPA-induced cellmigration. SIRNA-transfected SCC-9 cells were analyzed for transwellmigration toward fibronectin as chemoattractant. Each bar is the averageof quadruplicate values (mean±s.d.). *, P<0.001 for control siRNA+LPAvs. proAR siRNA+LPA. d, Effect of anti-AR neutralizing antibody andheparin on GPCR-induced EGFR and SHC tyrosine phosphorylation. SCC-9cells were pre-treated with anti-AR antibody (aAR Ab, 50 μg/mL, 60 min)or heparin (100 ng/mL, 15 min), and stimulated for 3′ min (EGFR) or 5min (SHC) as indicated. Precipitated EGFR and SHC were immunoblottedwith anti-phosphotyrosine antibody followed by reprobing of the samefilters with anti-EGFR and anti-SHC antibody, respectively. e,Association of Grb2 with SHC in vitro. SCC-9 cells were pre-incubatedwith inhibitors and stimulated for 5 min as indicated. Lysates wereincubated with GST-Grb-2 fusion protein or GST alone. Proteins wereimmunoblotted with monoclonal anti-SHC antibody. f, AR is required forGPCR-induced ERK/MAPK activation and Akt/PKB phosphorylation. SCC-9 orSCC-15 cells were pre-incubated with inhibitors and stimulated for 7′min. Phosphorylated ERK1/2 was detected by immunoblotting total lysateswith anti-phospho-ERK antibody. The same filters were re-probed withanti-ERK antibody. Quantitative analysis of ERK phosphorylation fromthree independent experiments (mean s.d.). *, P<0.05 for the differencebetween LPA vs. inhibitors+LPA. Stimulation of Akt/PKB. Cell lysateswere immunoblotted with anti-phospho-Akt/PKB antibody followed byreprobing of the same filters with anti-Akt/PKB antibody. g, Effect ofAR inhibition on LPA-induced DNA synthesis. SCC-15 cells were treatedwith inhibitors as indicated and incubated in the presence or absence ofligands (LPA; AR, 10 ng/ml) for 18 h. Cells were then pulse-labelledwith ³H-thymidine and thymidine incorporation was measured byliquid-scintillation counting. Quantitative analysis from threeindependent experiments (mean±s.d.). *, P<0.001 for LPA vs.inhibitors+LPA.

FIG. 3 Dominant negative TACE suppresses GPCR-induced AR release andEGFR signal transactivation. a, TACE is expressed in HNSCC cell lines.TACE was immunoprecipitated from lysates with monoclonal TACE/ADAM17antibody. HEK-293 cells transfected with human TACE cDNA served as apositive control. b, Timp-3 but not Timp-1 inhibits EGFR signaltransactivation. SCC-9 cells were infected with retrovirus encodinghuman Timp-1 or Timp-3. EGFR activation was determined by immunoblotafter stimulation with agonists as indicated (left panel). Expression ofTimp-1/3 carrying C-terminal VSV-tag was confirmed by immunoblottingtotal cell lysates with anti-VSV antibody (right panel). c, Expressionof wild type and dominant negative TACE or HA-tagged ADAM12 in SCC-9cells after retroviral gene transfer. Total lysates were immunoblottedas indicated. d, Dominant negative TACE abrogates LPA-induced proARcleavage (left panel) and AR release into cell culture medium (rightpanel) as determined by flow cytometric analysis and AR ELISA,respectively. e, Effect of dominant negative TACE on GPCR stimulatedEGFR signal transactivation.

FIG. 4 TACE siRNA inhibits EGFR signal transmission and cell migrationby GPCR agonists. a, TACE siRNA blocks endogenous TACE expression. SCC-9cells were transfected with TACE or ADAM12 siRNA. Gene expression wasanalyzed by RT-PCR (left panel) or immunoblot (right panel) withpolyconal anti-TACE antibody. b, Knockdown of TACE results inaccumulation of proAR at the cell surface. siRNA-transfected SCC-9 cellswere analyzed for AR cell surface content by FACS. c, EGFR signaltransmission upon GPCR activation requires TACE. SCC-9 cells weretransfected with siRNA and stimulated with agonists as indicated.Activation of EGFR, SHC, ERK and Akt was determined as described above.d, Squamous cancer cell motility in response to LPA depends on TACE.siRNA-transfected SCC-9 cells were treated with LPA or AR and analyzedin transwell migration assay.

FIG. 5 Immunoprecipitation of mature TACE protein by monoclonalantibodies raised against the metalloprotease-domain. HEK-293 cellstransiently expressing TACE-Hemagglutinin (HA) were serum-starved for 24h and lysed with TritonX-100 lysis buffer containing 5 μM BB94 asmetalloprotease inhibitor. 200 μg of crude lysate was used forimmunoprecipitation with 5 μg contol IgG (monoclonal anti-HA antibody)or 5 μg monoclonal anti-TACE antibody. Following SDS-polyacrylamide gelelectrophoresis, proteins were transferred to nitrocellulose membrane.Immunoprecipitated TACE protein was analysed by immunoblotting withpolyclonal TACE antibody (CHEMICON #19027).

FIG. 6 immunoprecipitation of endogenous TACE protein. SCC-9 cells wereserum-starved for 24 h and lysed with TritonX-100 lysis buffercontaining 5 μM BB94 as metalloprotease inhibitor. 200 μg of crudelysate was used for immunoprecipitation with 5 μg contol IgG (monoclonalanti-HA antibody) or 5 μg monoclonal anti-TACE antibody. FollowingSDS-polyacrylamide gel electrophoresis, proteins were transferred tonitrocellulose membrane. Immunoprecipitated TACE protein was analysed byimmunoblotting with TACE antibody (polyclonal antibody CHEMICON 19027).

FIG. 7 Flow cytometric analysis of TACE-binding of monoclonalantibodies. SCC9-cells were seeded, grown for 24 h. After collection,cells were stained with monoclonal TACE antibodies raised against themetalloprotease domain of TACE for 45 min. After washing withphosphate-buffered saline (PBS), cells were incubated with phycoerythrin(PE)-conjugated secondary antibodies for 45 min and washed again withPBS. Cells were analysed on a Becton Dickinson FACScalibur Fowcytometer.

FIG. 8 EGFR signal transactivation requires TACE activity. Serum-starvedSCC9 cells were preincubated for 30 minutes with 5 μg control IgG(monoclonal anti-HA antibody) or 5 μg monoclonal TACE antibody asindicated and treated with LPA (10 μM) for 3 min. After lysis, EGFR wasimmunoprecipitated (IP) using anti-EGFR antibody.Tyrosine-phosphorylated EGFR was detected by immunoblotting (IB) withanti-phosphotyrosine (αPY) antibody, followed by reprobing of the samefilter with anti-EGFR antibody.

FIG. 9 EGFR signal transactivation requires TACE activity. Serum-starvedSCC9 cells were preincubated for 30 minutes with 5 μg control IgG(monoclonal anti-HA antibody) or 5 μg monoclonal TACE antibody asindicated and treated with LPA (10 μM) for 3 min. After lysis, EGFR wasimmunoprecipitated (IP) using anti-EGFR antibody.Tyrosine-phosphorylated EGFR was detected by immunoblotting (IB) withanti-phosphotyrosine (αPY) antibody, followed by reprobing of the samefilter with anti-EGFR antibody.

EXAMPLE 1 EGFR Signal Transactivation in Squamous Cell CarcinomaRequires Proamphiregulin Cleavage by TACE

1. Methods

1.1 Cell Culture, Plasmids and Retroviral Infections

All cell lines (American Type Culture Collection, Manassas, Va.) wereroutinely grown according to the supplier's instructions. Transfectionsof HEK-293 cells were carried out by calcium phosphate coprecipitationas previously described (1). Anti-amphiregulin (AR), anti-HB-EGFneutralizing antibodies (R&D Systems, Minneapolis, Minn.), PTX, heparin(Sigma, St. Louis, Mo.), marimastat (BB2516, Sugen Inc., South SanFrancisco, Calif.), batimastat (BB94, British Biotech, Oxford, UK) wereadded to serum-starved cells before the respective growth factor.

Full-length cDNAs encoding ADAM10, 12, 15 and 17 were amplified by PCRfrom a human placenta cDNA library and subcloned into pcDNA3(Invitrogen, Carlsbad, Calif.) and pLXSN vectors (Clontech, Palo Alto,Calif.). For virus production dominant negative protease constructslacking the pro- and metalloprotease domains were generated as describedbefore (2,26). All protease constructs included a C-terminalhemagglutinin (HA) tag, detectable with an anti-HA monoclonal antibody(Babco, Richmond, Calif.). The amphotropic packaging cell line Phoenixwas transfected with pLXSN retroviral expression plasmids by the calciumphosphate/chloroquine method as described previously (29). At 24 h aftertransfection, the viral supernatant was collected and used to infectsubconfluent SCC-9 cells (5×104 cells/6-Well plate).

1.2 Protein Analysis

Cells were lysed and proteins immunoprecipitated as described (13).Western blots were performed according to standard methods. Theantibodies against human EGFR (108.1) and SHC (1), as well as a GST-Grb2fusion protein (5), have been characterized before. Phosphotyrosine wasdetected with the 4G10 monoclonal antibody (UBI, Lake Placid, N.Y.).Polyclonal anti-phospho-p44/p42 (Thr202/Tyr204) MAPK antibody andanti-phospho-Akt (Ser473) antibody were purchased from New EnglandBiolabs (Beverly, Mass.). Polyclonal anti-Akt1/2 and anti-ERK2 antibodywas from Santa Cruz Biotechnology (Santa Cruz, Calif.), anti-TACEantibodies from Chemicon (Harrow, UK).

1.3 Flow Cytometric Analysis and ELISA

ACS analysis was performed as described before (1). Cells were stainedwith ectodomain-specific antibodies against HB-EGF, AR (R&D Systems) orTGFa (Oncogene, Boston; MA). After washing with PBS, cells wereincubated with FITC-conjugated secondary antibody and analyzed on aBecton Dickinson FACScalibur flow cytometer.

Concentrations of free AR were determined by sandwich ELISA (R&DSystems) using monoclonal anti-AR capture antibody and biotinylatedpolyclonal detection antibody. Standards were recombinant human ARdiluted in culture medium. For statistical analysis Student's t-test wasused to compare data between two groups. Values are expressed asmean±s.d. of at least triplicate samples. P<0.05 was consideredstatistically significant.

1.4 RNA Interference and RT-PCR Analysis

Transfection of 21-nucleotide siRNA duplexes (Dharmacon Research,Lafayette, Colo., USA) for targeting endogenous genes was carried outusing Oligofectamine (Invitrogen) and 4.2 μg siRNA duplex per 6-wellplate as previously described (30). Transfected SCC-9 cells wereserum-starved and assayed 4 d after transfection. Highest efficienciesin silencing target genes were obtained by using mixtures of siRNAduplexes targeting different regions of the gene of interest. Sequencesof siRNA used were (SEQ ID NO:1) CCACAAAUACCUGGCUATAdTdT, (SEQ ID NO:2)AAAUCCAUGUAAUGCAGAAdTdT (AR); (SEQ ID NO:3) GUGAAGUUGGGCAUGACUAdTdT,(SEQ ID NO:4) UACAAGGACUUCUGCAUCCdTdT (HB-EGF); (SEQ ID NO:5)AACACUGUGAGUGGUGCCGdTdT, (SEQ ID NO:6) GAAGCAGGCCAUCACCGCCdTdT (TGFa);(SEQ ID NO:7) AAAGUUUGCUUGGCACACCUUdTdT, (SEQ ID NO:8)AAAGUAAGGCCCAGGAGUGUUdTdT, (SEQ ID NO:9) AACAUAGAGCCACUUUGGAGAdTdT(TACE); (SEQ ID NO:10) CCUCGCUGCAAAGAAUGUGdTdT (ADAM12), (SEQ ID NO:11)GACCUUGATACGACUGCUGdTdT (ADAM12); (SEQ ID NO:12) CGUACGCGGAAUACUUCGAdTdT(control, GL2).

Specific silencing of targeted genes was confirmed by Western blot(TACE) and RT-PCR analysis. RNA isolated using RNeasy Mini Kit (Qiagen,Hilden, Germany) was reverse transcribed using AMV Reverse Transcriptase(Roche, Mannheim, Germany). PuReTaq Ready-To-Go PCR Beads (AmershamBiosciences, Piscataway, N.J.) were used for PCR amplification. Customprimers (Sigma Ark, Steinheim, Germany) were proAR, (SEQ ID NO:13)5′-tggtgctgtcgctcttgata-3′ and (SEQ ID NO:14)5′-GCCAGGTATTTGTGGTTCGT-3′; proHB-EGF, (SEQ ID NO:15)5′-TTATCCTCCAAGCCACAAGC-3′ and (SEQ ID NO:16)5′-TGACCAGCAGACAGACAGATG-3′; proTGFa, (SEQ ID NO:17)5′-TGTTCGCTCTGGGTATTGTG-3′ and (SEQ ID NO:18)5′-ACTGTTTCTGAGTGGCAGCA-3′; TACE, (SEQ ID NO:19)5′-CGCATTCTCAAGTCTCCACA-3′ and (SEQ ID NO:20)5′-TATTTCCCTCCCTGGTCCTC-3′; ADAM12, (SEQ ID NO:21) 5′-CAGTTT CAC GGA AACCCA CT-3′ and (SEQ ID NO:22) 5′-GAC GAG AAC ACG TGC TGA GA-3′.PCR products were subjected to electrophoresis on a 2.5% agarose gel andDNA was visualized by ethidium bromide staining. Location of theproducts and their sizes were determined by using a 100-bp ladder(GIBCO, Gaithersburg, Md.) under ultraviolet illumination.1.5 Proliferation and Migration Assays

For the ³H-thymidine incorporation assay (5), SCC-15 cells were seededinto 12-well plates at 3×10⁴ cells/well. Upon serum deprivation for 48h, cells were subjected to pre-incubation and stimulation as indicated.After 18 h cells were pulse-labelled with ³H-thymidine (1 μCi/ml) for 4h, and thymidine incorporation was measured by trichloroacetic acidprecipitation and subsequent liquid-scintillation counting.

Analysis of cell motility was performed as described before (13) using amodified Boyden chamber. 24 h after transfection with siRNAs SCC-9 cellswere seeded into polycarbonate membrane inserts (6.5 mm diameter and 8μm pore size) In 24-transwell dishes at 1×10⁵ cells/well in the presenceor absence of agonist. The lower chamber was filled with standard mediumwithout FCS containing 10 μg/ml fibronectin as chemoattractant. Cellswere permitted to migrate for 36 h. Following incubation, nonmigratedcells were removed from the upper surface of the membranes. The cellsthat had migrated to the lower surface were fixed and stained withcrystal violet. The stained cells were solubilized in 10% acetic acid,absorbance at 570 nm was measured in a micro-plate reader.

2. Results

The GPCR-induced transactivation signal in HNSCC cells is sensitive tobroad-spectrum metalloprotease inhibitors such as batimastat (BB94) (13)and marimastat (BB2516; FIG. 1A). Consistent with a ligand-dependentmechanism of EGFR signal transactivation we found that the monoclonalanti-EGFR antibody ICR-3R which prevents binding of EGF-like growthfactors to the extracellular domain of the receptor (14) abrogated GPCR-and EGF-induced EGFR tyrosine phosphorylation in SCC-9 cells (FIG. 1A).In contrast, ICR-3R did not interfere with responses triggered bypervanadate, a potent tyrosine phosphatase inhibitor (15) whichincreases the tyrosine phosphorylation content of many intracellularproteins. Previous reports demonstrating that GPCR-induced EGFR tyrosinephosphorylation requires proteolytic cleavage of HB-EGF (1-3) promptedus to ask whether HB-EGF or other EGF-like growth factors are involvedin the EGFR transactivation pathway in head and neck cancer cells. BycDNA microarray analysis we found the expression of HB-EGF, TGFα and ARmRNAs in SCC-4, SCC-9, SCC-15 and SCC-25 cells (data not shown).Moreover, expression and cell surface localization of these ligands wereconfirmed by flow cytometry using ectodomain specific antibodies (FIG.1B, representative data shown for SCC-9). Surprisingly, treatment ofhead and neck cancer cells with LPA (10 μM) or the phorbol ester TPA (1mM), which acts as a general inductor of shedding events, reduced thecell surface content of endogenous proAR (FIG. 1C). However, in thiscellular context, LPA was not able to induce the proteolytic cleavage ofproTGFα or proHB-EGF, while stimulation with TPA resulted in ectodomaincleavage of both EGF-like growth factor precursors (data not shown).These findings suggested that LPA stimulation selectively inducesshedding of proAR in HNSCC. In addition, batimastat (10 μM) completelyabolished LPA-induced ectodomain cleavage of proAR (FIG. 1C) confirmingthe requirement of metalloprotease activity for proAR shedding. Inagreement with the observation that predominantly pertussis toxin(PTX)-sensitive G proteins of the Gi/o family are mediators ofLPA-induced EGFR tyrosine phosphorylation (FIG. 1A), PTX (100 ng/mL)partially inhibited proAR shedding at the cell surface of SCC-9 cells(FIG. 1C).

In addition to the decrease of cell-surface proAR, GPCR stimulationresulted in the accumulation of mature AR in cell culture medium asdetermined by sandwich-ELISA (FIG. 1D). The finding that AR release inresponse to carbachol was substantially lower compared to LPAstimulation suggested a direct correlation between the amount ofreleased AR and EGFR tyrosine phosphorylation content in response toGPCR ligands (FIG. 1A). Moreover, pre-incubation with batimastatcompletely prevented GPCR- and TPA-induced accumulation of AR in cellculture medium (FIG. 1D), confirming metalloprotease-dependency of ARrelease.

We used three approaches to determine if AR function is required forGPCR-induced EGFR tyrosine phosphorylation and downstream cellularresponses. First, we used small interfering RNA (siRNA) to silence theendogenous expression of proAR, proHB-EGF and proTGFα in SCC-9 cells.Effcient and specific knockdown of target gene expression was monitoredby RT-PCR (FIG. 2A) confirming that gene silencing occurred by mRNAdegradation. Concomitantly, the effect of siRNAs on the EGFRtransactivation signal was examined. As shown in FIG. 2B, siRNA to proARcompletely blocked GPCR-induced EGFR tyrosine phosphorylation. SiRNAs toproHB-EGF and proTGFα, however, did not significantly alter thetransactivation signal demonstrating specific requirement for proAR. Inaddition, we examined whether proAR knockdown affects the GPCR-inducedmotility of head and neck cancer cells. In fact, proAR siRNAsignificantly suppressed LPA-induced chemotactic migration in vitro(FIG. 2C).

Second, we examined the effect of AR neutralizing antibodies on EGFRtyrosine phosphorylation by LPA in the squamous cell carcinoma celllines SCC-4, SCC-9, SCC-15 and SCC-25. The results show thatpre-treatment with either a polyclonal goat or a monoclonal mouseantibody raised against the ectodomain of human AR inhibited the EGFRtransactivation signal (FIG. 2D, representative data shown for thepolyclonal anti-AR antibody in SCC-9 cells). Similar results wereobtained upon stimulation of head and neck cancer cells with carbachol(data not shown). In contrast, specific inhibition of HB-EGF by usingthe diphtheria toxin mutant CRM197 or anti-HB-EGF neutralizingantibodies showed no effect on LPA- or carbachol-induced EGFRtransactivation (data not shown).

Third, since AR contains a heparin-binding domain and theglycosaminoglycan heparin prevents AR-triggered mitogenic responses inkeratinocytes (16) and MCF-10A cells (17) we evaluated the effect ofheparin on the EGFR transactivation signal. As expected, heparin (100ng/mL) completely blocked EGFR tyrosine phosphorylation caused by LPA(FIG. 2D). Based on these findings we next examined whether AR functionis required for SHC activation downstream of the transactivated EGFR,since tyrosine phosphorylation of the adaptor protein SHC and formationof a SHC-Grb2-Sos complex is known to be a critical step in linking theactivated EGFR to the Ras/MAPK cascade (18). In fact, AR blockadecompletely prevented LPA-induced SHC tyrosine phosphorylation (FIG. 2D)and association with a glutathione-S-transferase (GST) Grb2 fusionprotein (FIG. 2E).

Several studies have previously demonstrated that EGFR transactivationis one important mechanism whereby GPCR agonists activate the ERK/MAPKpathway (4,12,19,20). To determine whether AR was required for LPAstimulated ERK/MAPK activation in HNSCC cells, the effect of ARinhibition on ERK1/2 activation was studied. As shown on FIG. 2F, ARneutralizing antibodies, heparin and batimastat prevented LPA-inducedERK activation in SCC-9 and SCC-15 cells. In addition to its mitogeniceffect, LPA can act as a survival factor by activating both the ERK/MAPKpathway and the phosphoinositide 3-kinase (PI3K)-dependentphosphorylation of Akt/PKB (21,22). We therefore raised the questionwhether LPA stimulation induces phosphorylation of Akt/PKB in head andneck cancer cells. The results indicate that LPA markedly increasedphosphorylation of Akt/PKB at Ser-473 (FIG. 2F). Moreover, Akt/PKBphosphorylation by LPA was sensitive to PI3K inhibition by wortmannin orLY294002 (data not shown) and was also abrogated by AR blockade orbatimastat treatment (FIG. 2F).

To further extend our studies on AR function for growth-promoting GPCRsignalling we assessed the effect of AR inhibition on LPA-induced DNAsynthesis. As shown in FIG. 2G, HNSCC cells displayed a significantreduction in the rate of DNA synthesis triggered by LPA upon ARinhibition suggesting that a full proliferative response by LPA requiresAR. Moreover, batimastat and the EGFR-specific inhibitor tyrphostinAG1478 decreased DNA synthesis by LPA to below basal level.Collectively, these data substantiate the requirement of AR for thegeneration of an EGFR-characteristic, mitogenic and motility-promotingtransactivation signal in HNSCC.

Recent observations have suggested a role of themetalloprotease-disintegrin TACE/ADAM17 in constitutive shedding ofproAR and other EGF-like growth factor precursors in mouse fibroblasts(23,24): Moreover, the proteolytic activity of TACE has been shown to beinhibited by the tissue inhibitor of metalloprotease-3 (Timp-3) but notTimp-1 in vitro (25). As TACE is widely expressed in HNSCC cell lines(FIG. 3A) we investigated the effect of Timp-1 and Timp-3 on the EGFRtransactivation signal. Indeed, ectopic expression of Timp-3 but notTimp-1 by retroviral transduction inhibited GPCR-induced EGFR tyrosinephosphorylation in SCC-9 cells (FIG. 3B). Furthermore, ectopicexpression of dominant negative TACE which lacks the pro- andmetalloprotease domain (26) (FIG. 3C) suppressed GPCR-induced proARcleavage, release of mature AR (FIG. 3D) and EGFR signal transactivationin SCC-9 cells (FIG. 3E). In contrast, neither dominant negative mutantsof ADAM10 (3) and ADAM12 (2) which have been shown to be involved inGPCR-triggered proHB-EGF processing nor an analogous ADAM15 mutantaffected the GPCR-induced responses (FIG. 3E, representative data shownfor ADAM12).

To independently verify the requirement of TACE for the EGFRtransactivation pathway in HNSCC we blocked endogenous expression ofTACE by RNA interference. Suppression of TACE expression was monitoredby RT-PCR and Western blot analysis (FIG. 4A). Interestingly,siRNA-directed inhibition of TACE resulted in the accumulation of proARat the cell surface of SCC-9 cells (FIG. 4B) supporting the view thatTACE is involved in basal proAR ectodomain processing. In addition, TACEsiRNA specifically suppressed GPCR-induced EGFR, SHC, ERK/MAPK andAkt/PKB activation (FIG. 4C). Finally, TACE siRNA also preventedmigration of SCC-9 cells in response to LPA (FIG. 4D).

3. Discussion

An increasing amount of experimental evidence supports the concept thatthe EGFR functions as a central integrator of diverse GPCR signals whichare thereby funnelled to downstream pathways (4,6,12). The datapresented here support an unexpected mechanism of EGFR transactivationin human cancer cells and identify a novel biological function for TACEin GPCR signalling. Our results demonstrate that GPCR-induced activationof TACE has biological consequences that can be attributed to anincrease in the amount of free AR. Other mechanisms, in whichHB-EGF-dependent transactivation of the EGFR is mediated by ADAM10 inlung epithelial cells (3) and COS-7 cells (27) or by ADAM12 incardiomyocytes (2) have been described. This is the first demonstration,however, that transmembrane proAR is cleaved in response to GPCRstimulation and also that AR is functionally relevant for mediatinghallmark cancer cell characteristics by GPCR agonists. We demonstratethat TACE-dependent AR release is a prerequisite to GPCR-induced EGFRstimulation, activation of the ERK/MAPK pathway, phosphorylation ofAkt/PKB, induction of cell proliferation and migration.

How TACE is activated by heterotrimeric G proteins is not known.Although ERK has been shown to bind to and phosphorylate the cytoplasmicdomain of TACE at threonine 735 in response to TPA stimulation (28),GPCR-induced AR release and EGFR tyrosine phosphorylation is insensitiveto MEK inhibitors in HNSCC cells (unpublished observation) suggestingERK not to be involved upstream of the EGFR. An important issue offuture studies will be to determine how GPCR signal transmission isdefined to be mediated by either ADAM10/HB-EGF, ADAM12/HB-EGF or TACE/ARmodules in a cell-type or physiology-dependent manner. Thus, ourexperimental results represent compelling evidence for the relevance ofphysiologically important GPCR ligands, TACE and AR in the mediation ofcritical cancer cell characteristics.

EXAMPLE 2 Production and Characterization of Monoclonal AntibodiesAgainst Tace

2.1 Generation of Monoclonal Antibodies

Monoclonal antibodies (Mabs) were raised against themetalloprotease-domain of human TACE (ADAM17). Recombinant protein wasused for immunization of BALB/c mice (J. H. Peters, H. Baumgarten and M.Schulze, Monoclonale Antikörper-Herstellung und Charakterisierung,Springer-Veriag, 1985, Berlin Heidelberg New York Tokio), Purificationof monoclonal antibodies took place with T-Gel™ Adsorbent from Pierce,Rockford, Ill., USA).

2.2 Functional Analysis

8 monoclonal antibodies recognizing the metalloprotease-domain of TACEwere identified by ELISA. These antibodies were used forimmunoprecipitation of lysates of HEK-293 cells transiently transfectedwith an eukaryotic expression plasmid encoding TACE tagged with thehemagglutinin epitope (TACE-HA) (31). The monoclonal antibodies 432-2,400-1, 343-3 and 432-7 specifically immunoprecipitate the mature form ofTACE, whereas the α-HA antibody immunoprecipitates predominantly thepro-form of TACE (FIG. 5).

Furthermore, the ability of the monoclonal antibodies toimmunoprecipitate endogenous TACE protein from lysates of SCC-9 cellswas tested. The MAbs 432-2, 400-1, 343-3 and 432-7 raised against themetalloprotease domain of TACE specifically immunoprecipitate the matureform of TACE and not the pro-form (FIG. 6).

Monoclonal antibodies were tested for their ability to detect TACE onthe cell surface of living cells. Antibodies 402-6 and 368-3 showed nocell surface staining, whereas 367-3 showed a weak signal. In contrast,antibodies 343-3, 374-5, 400-1, 432-2 and 432-7 showed a strong signal(FIG. 7).

Finally, we examined the effect of monoclonal TACE antibodies onLPA-induced EGFR tyrosine phosphorylation in the squamous cell carcinomacell line SCC-9. The results show that pre-treatment with 374-5, 432-2,400-1 and 367-3 inhibited the EGFR signal transactivation induced by LPA(FIG. 8), whereas direct stimulation of the EGFR with EGF was notaffected by pretreatment with monoclonal TACE antibodies (FIG. 9).

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1-19. (canceled)
 20. A method for modulating transactivation of receptor tyrosine kinases by G-protein or G protein-coupled receptor mediated signal transduction in a cell comprising specifically inhibiting the activity of amphiregulin and optionally TACE/ADAM17.
 21. The method of claim 20 wherein the cell is a human cell.
 22. The method of claim 20 wherein the cell is a carcinoma cell.
 23. The method of claim 22 wherein the cell is a squamous carcinoma cell.
 24. The method of claim 20 wherein the activity of amphiregulin and optionally TACE/ADAM17 is inhibited on the nucleic acid level.
 25. The method of claim 24 wherein the inhibition comprises a specific transcription inhibition.
 26. The method of claim 25 wherein the inhibition comprises application of anti-sense molecules, ribozymes or RNAi molecules directed against amphiregulin and optionally TACE/ADAM17 mRNA.
 27. The method of claim 24 wherein the inhibition comprises a gene inactivation.
 28. The method of claim 20 wherein the activity of amphiregulin and optionally TACE/ADAM17 is inhibited on the protein level.
 29. The method of claim 28 wherein the inhibition comprises a specific protein inhibition.
 30. The method of claim 28 wherein the inhibition comprises application of antibodies or antibody fragments directed against amphiregulin and optionally TACE/ADAM17.
 31. The method of claim 28 wherein the inhibition comprises application of low-molecular weight inhibitors of amphiregulin and optionally TACE/ADAM17.
 32. Use of a specific inhibitor of amphiregulin and optionally a specific inhibitor of TACE/ADAM17 for the manufacture of a medicament for the prevention and or treatment of a disorder which is caused by or associated with a transactivation of receptor tyrosine kinases by G protein or G protein-coupled receptor mediated signal transduction.
 33. The use of claim 32 wherein the disorder is a hyperproliferative disorder.
 34. The use of claim 33 wherein the disorder is cancer.
 35. The use of claim 34 wherein the disorder is squamous cell carcinoma.
 36. The use of 32 wherein the medicament additionally comprises pharmaceutically acceptable carriers, diluents and/or adjuvants.
 37. A method for the prevention and or treatment of a disorder which is caused by or associated with a transactivation of receptor tyrosine kinases by G protein or G protein-coupled receptor mediated signal transduction comprising administering a subject in need thereof an effective amount of a specific inhibitor of amphiregulin and optionally a specific inhibitor of TACE/ADAM17.
 38. A method for identifying modulators of receptor tyrosine kinase transactivation by G protein or G protein-coupled receptor mediated signal transduction, comprising determining, if a test compound is capable of specifically inhibiting the activity of amphiregulin and optionally the activity of TACE/ADAM17. 